Objective:
Develop at least one population of F2-derived soybean aphid clones from crossing two soybean aphid biotypes. Test derived aphid clones on plants with a specific Rag gene that differentiates the parental biotypes to determine potential inheritance of virulence on that gene; Create reciprocal symbiotic and aposymbiotic clones of each soybean aphid biotype and then test them for virulence on specific Rag genes to determine the potential effect of biotype-specific endosymbionts on virulence.

Approach:
Objective 1: Sexual forms of each soybean aphid biotype will be induced in controlled environmental chambers or naturally in the field inside field cages. Males and females (oviparae) of each biotype will be paired reciprocally in isolation inside plant cages in growth chambers, or field cages, allowed to mate and lay F1 hybrid eggs on buckthorn plants. Following a period of vernalization, F1 hybrid eggs will be induced to hatch inside growth chambers or naturally in field cages and F1 clones will be maintained in separate growth chambers. Sexual forms will then be induced from the F1 aphids as described above, allowed to mate and lay F2 eggs. Backcrosses (BC) will also be made between F1 hybrids and each biotype parent. F2 and BC eggs will be hatched and the fundatrices, or the aphids hatching from the eggs, will be collected and isolated individually to produce F2-derived clones. Each F2-derived and BC clone will be tested on the soybean plants with Rag genes that differentiate the biotype parents. Data on the proportion of clones that are virulent on the test resistant plants will be analyzed to determine the inheritance pattern of virulence.
Objective 2: Aposymbiotic clones of each of the three soybean aphid biotypes that are free of secondary endosymbionts will be established by means of injections with the antibiotic ampicillin, that does not harm Buchnera, into individual aphids for multiple consecutive generations. After antibiotic treatment, aposymbiotic clones will be maintained for three generations prior to being used for any subsequent experiment. Aphids will be tested for the presence of secondary endosymbionts at each generation using specific primers to the 16S-23S ribosomal RNA intergenic region. Sister clones without ampicillin treatment will also be maintained. Established aposymbiotic clones of each biotype will be reciprocally injected with secondary endosymbionts from the other biotypes using hemolymph, which is insect blood containing the endosymbiont, to establish additional sister clones of each soybean aphid biotype infected with secondary endosymbionts from different biotypes. All nine possible symbiont-biotype combinations will be produced.